Method for processing and preserving collagen-based tissues for transplantation

ABSTRACT

A method for processing and preserving an acellular collagen-based tissue matrix for transplantation is disclosed. The method includes the steps of processing biological tissues with a stabilizing solution to reduce procurement damage, treatment with a processing solution to remove cells, treatment with a cryoprotectant solution followed by freezing, drying, storage and rehydration under conditions that preclude functionally significant damage and reconstitution with viable cells.

RELATED APPLICATION

This application is a continuation-in-part of copending application Ser.No. 835,138 filed Feb. 12, 1992 which is a continuation-in-part ofcopending application Ser. No. 07/709,504 filed Jun. 3, 1991 which is acontinuation-in-part of application Ser. No. 07/581,584 filed Sep. 12,1990.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for procuring decellularizing andfurther processing and dry preserving collagen-based tissues derivedfrom humans and animals for transplantation into humans or otheranimals. These methods produce a tissue product that consists of aselectively preserved extracellular protein matrix that is devoid ofcertain viable cells which normally express major histocompatibilitycomplex antigenic determinants and other antigens which would berecognized as foreign by the recipient. This extracellular proteinmatrix is made up of collagen and other proteins and provides astructural template which may be repopulated with new viable cells thatwould not be rejected by the host. These viable cells may be derivedfrom the host (autologous cells) before or after transplantation or froman alternative human source including foreskin, umbilical cord oraborted fetal tissues. More particularly, this invention relates to theprocurement and processing of collagen-based tissues such thatcomplications following implantation (including but not limited toimmunorejection, contracture, calcification, occlusion, and infection)are significantly reduced relative to current implant procedures andmaterials.

2. Description of the Related Art

Tissue and organ transplantation is a rapidly growing therapeutic fieldas a result of improvements in surgical procedures, advancements inimmunosuppressive drugs and increased knowledge of graft/hostinteraction. Despite major advancements in this field, modern tissuetransplantation remains associated with complications includinginflammation, degradation, scarring, contracture, calcification(hardening), occlusion and rejection. There are numerous investigationsunderway directed toward the engineering of improved transplantabletissue grafts, however, it is generally believed in the industry thatideal implants have yet to be produced.

Autologous or self-derived human tissue is often used for transplantprocedures. These procedures include coronary and peripheral vascularbypass surgeries, where a blood vessel, usually a vein, is harvestedfrom some other area of the body and transplanted to correct obstructedblood flow through one or more critical arteries. Another application ofautologous tissue is in the treatment of third degree burns and otherfull-thickness skin injury. This treatment involves grafting of healthyskin from uninjured body sites to the site of the wound, a processcalled split-skin grafting. Additional applications of autologous tissuetransplantation include bone, cartilage and fascia grafting, used forreconstructive procedures.

The motive for using autologous tissue for transplantation is based uponthe concept that complications of immunorejection will be eliminated,resulting in enhanced conditions for graft survival. Unfortunately,however, other complications can ensue with autologous transplants. Forexample, significant damage can occur to several tissue components oftransplanted veins during harvesting and prior to implantation. Thisdamage can include mechanical contraction of the smooth muscle cells inthe vein wall leading to loss of endothelium and smooth muscle cellhypoxia and death. Hypoxic damage can result in the release of cellularlysosomes, enzymes which can cause significant damage to theextracellular matrix. Following implantation, such damage can lead toincreased platelet adhesion, leucocyte and macrophage infiltration andsubsequently further damage to the vessel wall. The end result of suchdamage is thrombosis and occlusion in the early post implant period.Even in the absence of such damage, transplanted autologous veinstypically undergo thickening of the vessel wall and advancingatherosclerosis leading to late occlusion. The exact cause of thisphenomena is uncertain but may relate to compliance mismatch of the veinin an arterial position of high blood pressure and flow rate. Thisphenomena may be augmented and accelerated by any initial smooth musclecell and matrix damage occurring during procurement. Occlusion oftransplanted veins can necessitate repeat bypass procedures, withsubsequent re-harvesting of additional autologous veins, or replacementwith synthetic conduits or non-autologous vessels.

Another example of complications resulting from autologous tissuetransplantation is the scarring and contracture that can occur withsplit-skin grafts for full-thickness wound repair. Split-skin grafts aretypically mechanically expanded by the use of a meshing instrument,which introduces a pattern of small slits in the skin. The split-skingraft is then stretched to cover a larger wound area. Dividing epidermalcells will ultimately grow into and cover the areas of the slits,however, the underlying dermal support matrix does not readily expandinto these areas. The dermal matrix, composed primarily of collagen,other extracellular protein matrix proteins, and basement membranecomplex, is responsible for the tensile, flexible nature of skin.Absence of a dermal matrix results in scarring and contracture in thearea of the slits. This contracture can be severe and in cases ofmassively burned patients that undergo extensive split-skin grafting,can necessitate subsequent release surgical procedures to restore jointmovement.

When the supply of transplantable autologous tissues is depleted, orwhen there is no suitable autologous tissue available for transplant(e.g., heart valve replacement), then substitutes may be used, includingman-made synthetic materials, animal-derived tissues and tissueproducts, or allogeneic human tissues donated from another individual(usually derived from cadavers). Man-made implant materials includesynthetic polymers (e.g. (PTFE) polytetrafluroethylene, Dacron andGoretex) sometimes formed into a tubular shape and used as a blood flowconduit for some peripheral arterial bypass procedures. Additionally,man-made synthetics (polyurethanes) and hydrocolloids or gels may beused as temporary wound dressings prior to split-skin grafting.

Other man-made materials include plastics and carbonized metals,fashioned into a prosthetic heart valve, utilized for aortic heart valvereplacement procedures. Synthetic materials can be made with lowimmunogenicity but are subject to other limitations. In the case ofmechanical heart valves, their hemodynamic characteristics necessitatelife-long anticoagulant therapy. Synthetic vascular conduits, often usedin above-the-knee peripheral vascular bypass procedures, are subjectedto an even higher incidence of occlusion than autologous grafts. In manycases, a preference is made for a biological implant which can be aprocessed animal tissue or a fresh or cryopreserved allogeneic humantissue.

Animal tissues (bovine or porcine) chemically treated are commonly usedas replacements for defective human heart valves, and have been used inthe past for vascular conduits. The concept in the chemical processingis to stabilize the structural protein and collagen matrix bycross-linking with glutaraldehyde or a similar cross-linking agent. Thistreatment also masks the antigenic determinants, such that the humanhost will not recognize the implant as foreign and precludes animmunorejection response. Glutaraldehyde-treated tissues, however, willnot allow in-migration of host cells which are necessary for remodeling,and will gradually harden as a result of calcification. For this reason,glutaraldehyde-treated tissues generally require replacement in 5-7years. Glutaraldehyde-treated bovine veins have been used in the pastfor vascular bypass bypass procedures, however, their use has beendiscontinued due to the unacceptable incidence of aneurysm formation andocclusion.

The use of allogeneic transplant tissues has been applied to heart valvereplacement procedures, arterial bypass procedures, bone, cartilage, andligament replacement procedures and to full-thickness wound treatment asa temporary dressing. The allogeneic tissue is used fresh, or may becryopreserved with the use of DMSO and/or glycerol, to maintainviability of cellular components. It is thought that the cellularcomponents contain histocompatibility antigens, and are capable ofeliciting an immune response from the host. In many cases, the patientreceiving the allogeneic transplant undergoes immunosuppressive therapy.Despite this therapy, many allogeneic transplants, including heartvalves and blood vessels, undergo an inflammatory response, and failwithin 5-10 years. Allogeneic skin is typically rejected within 1-5weeks of application, and has never been demonstrated to be permanentlyaccepted by the host, even with the use of immunosuppressive drugs.

Alternative processing methods have been developed by others that areintended to address the limitations of allogeneic and animal-derivedtransplant tissues. Freeze-drying is used routinely in the processing ofallogeneic bone for transplantation. It has been found that the freezedrying process results in a graft which elicits no significant rejectionresponse as compared to fresh or cryopreserved allogeneic bone. Thefreeze-dried bone following implant acts as a template, which issubsequently remodelled by the host. When the freeze-drying process hasbeen applied to more complex tissues such as heart valves, the resultshave been mixed but overall unsatisfactory. A study was conducted inwhich 15 allogeneic heart valves were processed by freeze-drying priorto transplantation. Most of the freeze-dried valves failed due tomechanical causes in the early post-graft interval. Those freeze-driedvalves which did not fail, however, demonstrated prolonged functionality(up to 15 years).

Enzymes and detergent processing has also been used to remove antigeniccells from collagen-based transplantable tissues. Organic solvents anddetergent treatments have been used successfully with relatively simpletissues such as dura mater used in reconstructive surgical procedures.Chemical processing of more complex structures such as heart valves,vascular conduits and skin, however, has had only limited success inclinical applications.

The invention of this patent is a comprehensive processing techniquethat addresses potential damaging events in the preparation of complexcollagen-based tissues for transplantation. The technology combines bothbiochemical and physical processing steps to achieve the ideal featuresof template function such that the tissue graft can be remodeled forlong-term maintenance by the host.

BRIEF SUMMARY OF THE INVENTION

In its preferred form, the method of this invention includes the stepsof processing biological tissues including treatment with a stabilizingsolution to reduce procurement damage, treatment with a processingsolution to remove cells and other antigenic tissue components,treatment with a cryoprotectant solution, freezing and storage underspecific conditions to avoid functionally significant damaging icecrystal formation, drying under conditions to prevent damaging icerecrystallization, storage in the dry state at above freezingtemperatures, rehydration under specific conditions and with arehydration solution to minimize surface tension damage and furtheraugment the selective preservation of the matrix, and reconstitutionwith viable cells that will not be rejected by the host.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method for processing and preservingcollagen-based biological tissues for transplantation, through steps ofchemical pretreatment and cell removal, cryopreparation, drystabilization, drying, rehydration and cellular reconstitution. Theprocessing and preservation method is designed to generate atransplantable biological tissue graft that specifically meets thefollowing criteria:

-   -   (a) provides an extracellular protein and collagen matrix which        can be remodelled and repaired by the host,    -   (b) provides an intact basement membrane for secure reattachment        of viable endothelial or epithelial cells,    -   (c) does not elicit an immune response by the host,    -   (d) does not calcify, and    -   (e) can be easily stored and transported at ambient        temperatures.

In the preferred embodiment, the biological tissue to be processed isfirst procured or harvested from a human cadaver or animal donor andimmediately placed in a stabilizing transportation solution whicharrests and prevents osmotic, hypoxic, autolytic and proteolyticdegradation, protects against bacterial contamination and reducesmechanical damage that can occur with tissues that contain smooth musclecomponents (e.g. blood vessels). The stabilizing solution generallycontains an appropriate buffer, one or more antioxidants, one or moreoncotic agents, an antibiotic, one or more protease inhibitors, and insome cases, a smooth muscle relaxant.

In the preferred embodiment, the tissue is then incubated in aprocessing solution to remove viable antigenic cells (includingepithelial cells, endothelial cells, smooth muscle cells andfibroblasts) from the structural matrix without damaging the basementmembrane complex or the structural integrity of the collagen matrix. Theprocessing solution generally contains an appropriate buffer, salt, anantibiotic, one or more detergents, one or more protease inhibitors,and/or one or more enzymes. Treatment of the tissue with this processingsolution must be at a concentration for a time duration such thatdegradation of the basement membrane complex is avoided and thestructural integrity of the matrix is maintained including collagenfibers and elastin.

After the tissue is decellularized, it is preferably incubated in acryopreservation solution. In the preferred embodiment, this solutiongenerally contains one or more cryoprotectants to minimize ice crystaldamage to the structural matrix that could occur during freezing, andone or more dry-protective components, to minimize structural damagealteration during drying and may include a combination of an organicsolvent and water which undergoes neither expansion or contractionduring freezing. As an alternate method, the decellularized tissuematrix can be fixed with a crosslinking agent such as glutaraldehyde andstored prior to transplantation. Following incubation in thiscryopreservation solution, the tissue is packaged inside a sterilecontainer, such as a glass vial or a pouch, which is permeable to watervapor yet impermeable to bacteria.

In the preferred embodiment, one side of this pouch consists of medicalgrade porous Tyvek membrane, a trademarked product of DuPont Company ofWilmington, Del. This membrane is porous to water vapor and imperviousto bacteria and dust. The Tyvek membrane is heat sealed to a 2.5millimeter impermeable polythylene laminate sheet, leaving one sideopen, thus forming a two-sided pouch. The open pouch is sterilized bygamma radiation prior to use. The tissue is aseptically placed throughthis opening into the sterile pouch. The open side is then asepticallyheat sealed to close the pouch. The packaged tissue is henceforthprotected from microbial contamination throughout subsequent processingsteps.

In the preferred embodiment, the packaged tissue is cooled to a lowtemperature at a specified rate which is compatible with the specificcryoprotectant to minimize damaging hexagonal ice and to generate theless stable ice forms of amorphous and cubic phases. The tissue is thendried at a low temperature under vacuum conditions, such that watervapor is removed sequentially from each ice crystal phase without icerecrystallization. Such drying is achieved either by conventional freezedrying or by using a previously patented molecular distillation dryer.Suitable molecular distillation dryers can be obtained from LifeCellCorporation in the Woodlands, Tex. and are disclosed in U.S. Pat. Nos.4,567,847 and 4,799,361 which are incorporated herein by reference.

At the completion of the drying cycle of samples dried in a pouch, thevacuum of the freeze drying apparatus is reversed with a dry inert gassuch as nitrogen, helium or argon. While being maintained in the samegaseous environment, the semipermeable pouch is placed inside animpervious pouch which is further heat or pressure sealed. The finalconfiguration of the dry sample is therefore in an inert gaseousatmosphere, hermetically sealed in an impermeable pouch.

At the completion of the drying cycle of samples dried in a glass vial,the vial is sealed under vacuum with an appropriate inert stopper andthe vacuum of the drying apparatus reversed with an inert gas prior tounloading.

In the preferred embodiment, the packaged dried tissue may be stored forextended time periods under ambient conditions. Transportation may beaccomplished via standard carriers and under standard conditionsrelative to normal temperature exposure and delivery times.

In the preferred embodiment, the dried tissue is rehydrated prior totransplantation. It is important to minimize osmotic forces and surfacetension effects during rehydration. The aim in rehydration is to augmentthe selective preservation of the extracellular support matrix whileremoving any residual antigenic cells and other potentially antigeniccomponents. Appropriate rehydration may be accomplished by an initialincubation of the dried tissue in an environment of about 100% relativehumidity, followed by immersion in a suitable rehydration solution.Alternatively, the dried tissue may be directly immersed in therehydration solution without prior incubation in a high humidityenvironment. Rehydration should not cause osmotic damage to the sample.Vapor rehydration should ideally achieve a residual moisture level of atleast 15% and fluid rehydration should result in a tissue moisture levelof between 20% and 70%.

Depending on the tissue to be rehydrated, the rehydration solution maybe simply normal saline, Ringer's lactate or a standard cell culturemedium. Where the tissue is subject to the action of endogenouscollagenases, elastases or residual autolytic activity from previouslyremoved cells, additives to the rehydration solution are made andinclude protease inhibitors. Where residual free radical activity ispresent, agents to protect against hypoxia are used includingantioxidants, enzymatic agents which protect against free radical damageand agents which minimize the disturbance of biochemical pathways whichresult from hypoxic damage. Antibiotics may also be included to inhibitbacterial contamination. Oncotic agents being in the form ofproteoglycans, dextran and/or amino acids may also be included toprevent osmotic damage to the matrix during rehydration. Rehydration ofa dry sample is especially suited to this process as it allows rapid anduniform distribution of the components of the rehydration solution. Inaddition, the rehydration solution may contain specific components notused previously, for example diphosphonates to inhibit alkalinephosphatase and prevent subsequent calcification. Agents may also beincluded in the rehydration solution to stimulate neovascularization andhost cell infiltration following transplantation of the rehydratedextracellular matrix. Alternatively, rehydration may be performed in asolution containing a crosslinking agent such as glutaraldehyde

Immunotolerable viable cells must be restored to the rehydratedstructural matrix to produce a permanently accepted graft that may beremodeled by the host. In the preferred embodiment, immunotolerableviable cells may be reconstituted by in vitro cell culturing techniquesprior to transplantation, or by in vivo repopulation followingtransplantation.

In the preferred embodiment the cell types used for in vitroreconstitution will depend on the nature of the transplantable graft.The primary requirement for reconstitution of full-thickness skin fromprocessed and rehydrated dermis is the restoration of epidermal cells orkeratinocytes. These cells may be derived from the intended recipientpatient, in the form of a small meshed split-skin graft or as isolatedkeratinocytes expanded to sheets under cell culture conditions.Alternatively, allogeneic keratinocytes derived from foreskin or fetalorigin, may be used to culture and reconstitute the epidermis.

The important cell for reconstitution of heart valves and vascularconduits is the endothelial cell, which lines the inner surface of thetissue. Endothelial cells may also be expanded in culture, and may bederived directly from the intended recipient patient or from umbilicalarteries or veins.

Following drying, or following drying and rehydration, or followingdrying, rehydration and reconstitution, the processed tissue graft willbe transported to the appropriate hospital or treatment facility. Thechoice of the final composition of the product will be dependent on thespecific intended clinical application.

In the practice of this invention, it is fundamental that suitabletissues are obtained prior to processing. Human cadaver tissues areobtainable through approximately 100 tissue banks throughout the nation.Additionally, human tissues are obtainable directly from hospitals. Asigned informed consent document is required from the donor's family toallow harvesting of tissues for transplantation. Animal tissues areobtainable from a number of meat processing companies and from suppliersof laboratory animals. The particular type of tissue harvested is notlimiting on the method of this invention. However, processing of thetissue is enhanced by the use of specific procurement procedures, andtreatment with a stabilizing solution to prevent certain mechanical andbiochemical damaging events.

The harvested tissues can undergo a variety of mechanical andbiochemical damaging events during procurement. Both the cellularcomponents and the extracellular matrix can be injured during theseevents. Damage to the extracellular matrix occurs primarily as a resultof destabilization of the cellular component. The intent of thisinvention is to ultimately remove this cellular component and tooptimally preserve the extracellular matrix, therefore the stabilizingsolution is formulated to minimize the initial cellular and subsequentlythe extracellular matrix damage. The extracellular protein and collagenmatrix comprises a native three dimensional lattice that includesvarious proteins such as Type I collagen, Type II collagen, Type IIIcollagen, Type IV collagen, elastin, laminin, teninsin and actinin, andproteoglycans.

The initiating event in cellular damage is hypoxia (deficiency of oxygenreaching tissues of the body) and a lack of nutrient supply required forthe cell to maintain metabolism and energy production. Hypoxia andespecially hypoxia and reperfusion results in the generation of freeradicals such as hydrogen peroxide, an oxidizing species that reactswith cellular components including membranes and proteins. Thesubsequent changes of lipid peroxidation and crosslinking result instructural and functional derangement of the cell and initiate releaseof autolytic enzymes (normally contained in lysosomes) into theextracellular matrix. The damage to the matrix is two-fold, oxidantdamage and enzymatic degradation. A lack of nutrient supply amplifiesthese events in that the cell can no longer provide the energyrequirements necessary to maintain its defense mechanisms againsthypoxic damage. In minimizing these events, several approaches arepossible. These include the use of enzymes (superoxide dismutase andcatalase) to neutralize the superoxide anion and hydrogen peroxide orcompounds which can directly react with and neutralize otherfree-radical species. These compounds referred to as antioxidantsinclude tertiary butylhydroquinone (BHT), alpha tocopherol, mannitol,hydroxyurea, glutathione, ascorbate, ethylenediaminetetraacetic acid(EDTA) and the amino acids histidine, proline and cysteine. In additionto antioxidants, the stabilizing solution generally contains agents toinhibit hypoxic alteration to normal biochemical pathways, for example,allopurinol to inhibit xanthine dehydrogenase, lipoxigenase inhibitors,calcium channel blocking drugs, calcium binding agents, iron bindingagents, metabolic intermediaries and substrates of adenosinetriphosphate (ATP) generation.

The stabilizing solution also generally contains one or moreantibiotics, antifungal agents, protease inhibitors, proteoglycans, andan appropriate buffer. Antibiotics are necessary to inhibit or preventbacterial growth and subsequent tissue infection. Antibiotics may beselected from the group of penicillin, streptomycin, gentamicinkanamycin, neomycin, bacitracin, and vancomycin. Additionally,anti-fungal agents may be employed, including amphotericin-B, nystatinand polymyxin.

Protease inhibitors are included in the stabilizing solution to inhibitendogenous proteolytic enzymes which, when released, can causeirreversible degradation of the extracellular matrix, as well as therelease of chemoattractant factors. These chemoattractants solicit theinvolvement of polymorphonuclear leukocytes, macrophages and otherkiller cells which generate a nonspecific immune response that canfurther damage the extracellular matrix. Protease inhibitors areselected from the group consisting of N-ethylmaleimide (NEM),phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid(EDTA), ethylene glycol-bis (2-aminoethyl ether)-N,N,N′,N′-tetraaceticacid (EGTA), leupeptin, ammonium chloride, elevated pH and apoprotinin.

Proteoglycans are included in the stabilizing solution to provide acolloid osmotic balance between the solution and the tissue, therebypreventing the diffusion of endogenous proteoglycans from the tissue tothe solution. Endogenous proteoglycans serve a variety of functions incollagen-based connective tissue physiology. They may be involved in theregulation of cell growth and differentiation (e.g. heparin sulfate andsmooth muscle cells) or, alternatively, they are important in preventingpathological calcification (as with heart valves). Proteoglycans arealso involved in the complex regulation of collagen and elastinsynthesis and remodelling, which is fundamental to connective tissuefunction. Proteoglycans are selected from the group of chondroitinsulfate, heparin sulfate, and dermatan sulfate. Non-proteoglycan asmoticagents which may also be included are polymers such as dextran andpolyvinyl pyrolodone (PVP) and amino acids such as glycine and proline.

The stabilizing solution also generally contains an appropriate buffer.The nature of the buffer is important in several aspects of theprocessing technique. Crystalloid, low osmotic strength buffers havebeen associated with damage occurring during saphenous vein procurementand with corneal storage. Optimum pH and buffering capacity against theproducts of hypoxia damage (described below), is essential. In thiscontext the organic and bicarbonate buffers have distinct advantages.(In red cell storage, acetate-citrate buffers with glycine and glucosehave been shown to be effective in prolonging shelf-life and maintainingcellular integrity.) The inventors prefer to use an organic bufferselected from the group consisting of 2-(N-morpholino)ethanesulfonicacid (MES), 3-(N-morpholine)propanesulfonic acid (MPOS) andN-2-hydroxyethylpiperazine-N′-2-ethane-sulfonic acid (HEPES).Alternatively, a low salt or physiological buffer, including phosphate,bicarbonate and acetate-citrate, may be more appropriate in certainapplications.

In another preferred embodiment, components of the stabilizing solutionaddress one or more of the events that occur during the harvesting ofvascular tissues, such as spasm, hypoxia, hypoxia reperfusion, lysosomalenzyme release, platelet adhesion, sterility and buffering conditions.Involuntary contraction of the smooth muscle lining of a blood vesselwall can result from mechanical stretching or distension, as well asfrom the chemical action of certain endothelial cell derived contractionfactors, typically released under hypoxic (low oxygen) conditions. Thisinvoluntary contraction results in irreversible damage to the muscleitself, the endothelial cells and the surrounding extracellular matrix.For this reason, the stabilizing solution for blood vessels includes oneor more smooth muscle relaxants, selected from the group of calcitoningene related peptide (CGRP), papaverine, sodium nitroprusside (NaNP), H7(a protein Kinase C inhibitor) calcium channel blockers, calciumchelators, isoproterenol, phentolamine, pinacidil,isobutylmethylxanthine (IBMX), nifedepine and flurazine. The harvestedtissue is immediately placed into this stabilizing solution and ismaintained at 4° C. during transportation and any storage prior tofurther processing.

In the practice of this invention, it is essential that the harvestedtissue be processed to remove antigenic cellular components.

Decellularization can be accomplished using a number of chemicaltreatments, including incubation in certain salts, detergents orenzymes. The use of the detergent Triton X-100, a trademarked product ofRohm and Haas Company of Philadelphia, Pa., has been demonstrated toremove cellular membranes, as detailed in U.S. Pat. No. 4,801,299. Otheracceptable decellularizing detergents include polyoxyethylene (20)sorbitan mono-oleate and polyoxyethylene (80) sorbitan mono-oleate(Tween 20 and 80), sodium deoxycholate,3-[(3-chloramidopropyl)-dimethylammino]-1-propane-sulfonate,octyl-glucoside and sodium dodecyl sulfate.

Alternatively, enzymes may be used to accomplish decellularization,including but not limited to dispase II, trypsin, and thermolysin. Theseenzymes react with different components of collagen and intercellularconnections in achieving their effects. Dispase II attacks Type IVcollagen, which is a component of the lamina densa and anchoring fibrilsof the basement membrane. Thermolysin attacks the bulbous phemphigoidantigen in the hemidesmosome of the basal layer of keratinocytes.Trypsin attacks the desmosome complex between cells. Due to theproteolytic nature of these enzymes, care must be taken that cellularremoval occurs without significant damage to the extracellular matrix,including the basement membrane complex. This is a function ofconcentration, time and temperature. If used for too long a time or attoo high a concentration, dispase II for example can completely removethe basement membrane complex from the dermis.

For example, with human cadaver skin Dispase II at 1.0 units/ml for 90minutes at 37° C. will remove all heratinocytes except the basal layer,while some damage is already occurring to the basement membrane complex.Thermolysin at 200 ug/ml for 30 minutes at 4° C. will essentially removeall keratinocytes without damage to the basement membrane complex onsome occasions, but this varies from donor to donor with evidence ofbasement membrane damage being seen in some donors. Incubation of skinin 1 molar sodium chloride for 16 hours for human skin and 48 hours forporcine skin will routinely allow clean separation of the epidermis anddermis without damage to the basement membrane complex.

In addition to salts, detergents and enzymes, the processing solutionalso contains certain protease inhibitors, to prevent degradation of theextracellular matrix. Collagen-based connective tissues containproteases and collagenases as endogenous enzymes in the extracellularprotein matrix. Additionally, certain cell types including smooth musclecells, fibroblasts and endothelial cells contain a number of theseenzymes inside vesicles called lysosomes. When these cells are damagedby events such as hypoxia, the lysosomes are ruptured and their contentsreleased. As a result, the extracellular matrix can undergo severedamage from protein, proteoglycan and collagen breakdown. This damagemay be severe, as evidenced in clinical cases of cardiac ischemia wherea reduction in oxygen which is insufficient to cause cell death resultsin pronounced damage to the collagen matrix. Additionally, a consequenceof extracellular breakdown is the release of chemoattractants, whichsolicit inflammatory cells, including polymorphonuclear leukocytes andmacrophages, to the graft, which are intended to remove dead or damagedtissue. These cells also, however, perpetuate the extracellular matrixdestruction through a nonspecific inflammatory response. Accordingly,the processing solution contains one or more protease inhibitorsselected from the group of N-ethylmaleimide (NEM),phenylmethylsulfonylfluoride (PMSF) ethylenediamine tetraacetic acie(EDTA), ethylene glycol-bis-(2-aminoethyl(ether)NNN′N′-tetraacetic acid,ammonium chloride, elevated pH, apoprotinin and leupeptin to preventsuch damage.

In addition to salts, detergents, enzymes and protease inhibitors, theprocessing solution generally contains an appropriate buffer. This mayinvolve one of many different organic buffers which are described above.The inventors prefer to use an organic buffer selected from the groupconsisting of 2-(N-morpholino)ethanesulfonic acid (MES), Tris(hydroxymethyl)amionomethane (TRIS) and(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES).Alternatively, a low salt or physiological buffer including phosphatebicarbonate acetate citrate glutamate with or without glycine, may bemore appropriate in certain applications. Low salt or physiologicalbuffers are more able to support the infiltration of the graft withviable cells and hence are more relevant when cellular infiltrationincluding neovascularization is essential to early survival of the graftas in transplated dermal matrix.

As the processing solution may contain chemicals that would beirritating or inflammatory on transplantation, it is important to thepractice of this invention that the processing solution be thoroughlyrinsed from the tissue. In the preferred embodiment, this washing occursby rinsing in sufficient changes of appropriate buffer, until residuesof the processing solution are reduced to levels compatible withtransplantation. Alternatively, components of the processing solutionmay be neutralized by specific inhibitors, e.g., dispase II byethylenediaminetetraacetic acid (EDTA) or trypsin by serum.

The cryopreparation or freezing of the tissue takes place followingthorough washing. Biological materials generally undergo significantdeterioration during freezing and thawing or following freeze-drying byconventional means. Accordingly, these steps should be avoided prior toincubation of the processed, decellularized tissue in a cryoprotectantsolution by the method described in this application.

The initial steps of cryopreserving the decellularized tissue includesincubating the tissue in a cryosolution prior to the freezing step. Thecryosolution comprises an appropriate buffer, one or morecryoprotectants and/or dry protectants with or without an organicsolvent which in combination with water undergoes neither expansion orcontraction.

An appropriate buffer may involve any of the previously describedbuffers utilized in procurement or decellularization processing of theharvested tissue.

In addition to an appropriate buffer, the cryosolution generallycontains a cryoprotectant. Cryoprotectants raise the glass transitiontemperature range of the tissue thereby allowing optimum stabilizationof the tissue in the frozen state. By raising this range, the tissue canbe dried at a faster rate. The cryoprotectant also decreases iceformation for a given cooling rate allowing to some degree vitrification(absence of a crystalline lattice), but to a greater extent, theformation of cubic ice. With current methods of ultra-rapid cooling inthe absence of cryoprotectants, vitrification is only achieved with verysmall samples, and only to a depth of a few microns. Cubic and hexagonalice are then encountered. Vitrified water and cubic ice are lessdamaging to extracellular matrix components than is hexagonal ice. Insome cases, however, it is permissible to allow hexagonal ice to occur(e.g., the processing of skin). Some degree of hexagonal ice formationis permissable when it does not result in impairment of the functionalcharacteristics of the tissue. Heart valves following implantation aresubject to repetitive stress and hence will tolerate less ice crystaldamage than, for example, dermis.

Various cryoprotectants can be used in the present invention. Theseinclude: dimethylsulfoxide (DMSO), dextran, sucrose, 1,2 propanediol,glycerol, sorbitol, fructose, trehalose, raffinose, propylene glycol,2-3 butane diol, hydroxyethyl starch, polyvinylpyrrolidone (PVP),proline (or other protein stabilizers), human serum albumin andcombinations thereof. Suitable cryoprotectants structure water moleculessuch that the freezing point is reduced and/or the rate of coolingnecessary to achieve the vitreous phase is reduced. They also raise theglass transition temperature range of the vitreous state.

The cryosolution may also include exposing the biological tissue to oneor more dry protectant compounds. Dry protectants, by definition,stabilize samples in the dry state. Some cryoprotectants also act as dryprotectants. Some compounds possess variable amounts of each activity,e.g., trehalose is predominantly a dry protectant and a weakercryoprotectant, whereas sucrose is predominantly a cryoprotectant and aweaker dry protectant. For example, trehalose and polyhydroxylcarbohydrates bind to and stabilize macromolecules such as proteins.Various dry protectants can be used in the present invention: sucrose,raffinose, trehalose, zinc, proline (or other protein stabilizers),myristic acid, spermine (a polyanionic compound) and combinationsthereof.

The combination of 0.5 Molar dimethyl sulfoxide, 0.5 M propylene glycol0.25 M 2-3 butanediol, 1.0 M proline, 2.5% raffinose 15%polyvinylpyrrolidone and 15% dextran (MWT 70,000) in combination withcooling rates of the order of −2500° C./second has been shown to beeffective in maintaining the structural integrity of human saphenousveins following both freezing and drying. The inventors have alsodemonstrated that when this solution of cryoprotectants, dry protectantsand buffer are used with a larger tissue sample, such as a heart valve,then the tissue can undergo cracking following freezing and/or drying.This phenomena can be overcome by replacing a percent of the water withan organic solvent such as formamide. The percent (50%) is determined asthat combination of solvent, water, cryoprotectants and dry protectantswhich will not expand or contract during freezing. Formamide (HCONH₂) isa one carbon, hydrophilic, organic solvent which dissolves carbohydratebased cryoprotectants. It may be substituted with other organic solventswith similar properties such as dimethylformamide, dimethylsulfoxide(DMSO), glycerol, proplyene glycol, ethylene glycol, and pyridine.

The biological samples are incubated in the cryosolutions for a periodof a few minutes to a few hours before they are rapidly cooled. Ingeneral, cryopreservation is performed as a continuous sequence ofevents. The tissue is first incubated in the cryosolution for a definedperiod (0.5 to 2 hours) until complete penetration of the components ofthe cryosolution is achieved and the sample is then frozen to atemperature at which it is stable, usually less than −20° C.

The inventors have been involved in the development of cryofixation andultralow temperature molecular distillation drying as a method forpreparing biological samples for electron microscopic analysis. Tovalidate this approach, they investigated the relationship betweendrying characteristics and ice phases present within frozen samples.

Sample preparation for electron microscopy by purely physical or dryprocessing techniques has theoretical appeal, especially when theultimate aim is the analysis of both ultrastructure and biochemistry.Since the earliest days of electron microscopy, several attempts havebeen made to refine and develop freezing and vacuum drying or thefreeze-drying (FD) process for cell and tissue samples.

Despite the conceptual advantages and the progress made, freeze-dryingfor electron microscopy has yet to achieve the status of a routine,broadly applicable technique. Several reasons account for this. First,the ultrastructural preservation is often inferior when compared toconventional chemical, or wet processing techniques or hybrid techniquessuch as freeze substitution. Second, there are numerous practicalproblems with sample manipulation, temperature control, vacuumparameters, and end processing protocols. Third, and perhaps mostfundamentally, is a belief that drying at temperatures below −123° C. toavoid ultrastructural damage is either impossible or impractical. As aresult of these practical and theoretical obstacles, only sporadicinvestigation of low temperature freeze-drying has been reported.

The basis of this theoretical barrier comes from application of thekinetic gas theory and the predicted sublimation rates as expressed bythe Knudsen equation:${Js} = {{NPs}( \frac{M}{2\quad\pi\quad{QT}} )}^{0.5}$where

-   -   Js=sublimation rate    -   N=coefficient of evaporation    -   Ps=saturation vapor pressure    -   Q=universal gas constant    -   T=absolute temperature of the sample    -   M=molecular weight of water.

For theoretically ideal drying conditions, this equation states that thesublimation rate is directly proportional to the saturation vaporpressure of water within the sample and inversely proportional to theabsolute temperature of the sample. Although the temperature of thesample is clearly definable, saturation vapor pressure is a more complexparameter.

Prior applications of this equation have used saturation vapor pressureswhich were theoretically determined. These theoretical vapor pressures,however, include the latent heat of fusion, and hence, are applicableonly to hexagonal ice. Calculations based on these theoretical valueshave led to conclusions such as “at 150K it would take 3.5 years untilan ice layer of 1 mm thickness is completely removed by freeze drying.It is therefore unrealistic to attempt freeze drying at temperaturesbelow 170K.”

Several phases of ice other than hexagonal, however, can coexist withina sample depending upon the mode of cooling and the use ofcryoprotectants. These different phases can be achieved by severalmethods including; vapor condensation, hyperbaric application andultrarapid quench cooling.

The major phases of ice now recognized are amorphous, cubic, andhexagonal. These ice phases exhibit different stabilities, which wouldsuggest that the saturation vapor pressures would also be different. Ithas been determined that for vapor condensed water at temperatures whereboth phases can coexist, the saturation vapor pressure of amorphous iceis one to two logs higher than that of cubic ice.

Application of these experimentally determined saturation vaporpressures in the Knudsen equation reduces the drying time at 150K from3.5 years to 0.035 years, or 12.7 days, for 1 mm of amorphous ice.Because quench cooling techniques of biological samples achieveapproximately 5 μm of this phase, the drying time of this component,based solely on the Knudsen equation, would be of the order of 1.5hours. Hence, in terms of practical drying times, the theoreticalbarrier to drying at ultralow temperatures can be overcome.

Drying, however, is not a static, but a rate-dependent process. Inaddition to saturation vapor pressure of the different ice phases, onemust also account for the rate of transition from one phase to anotherwith increasing temperature. For electron microscopy sample preparation,drying should ideally occur without any such transition ordevitrification. Information as to the rate of these transitions islimited. It has been found that the amorphous to cubic transitionoccurred as an irreversible process strongly dependent upon temperaturein the range of −160° C. to −130° C. and expressed byt=2.04×10²⁸×exp(−0.465T)The cubic to hexagonal transition was less temperature-dependent,occurring in the range of −120° C. to −65° C., and expressed byt=2.58×10¹²×exp(−0.126T)Interestingly, when the sample temperature was increased at a rate of 5°C./minute, the amorphous to cubic transition occurred as a sudden eventnear −130° C.

Based upon the above data, the transition rate, as well as thesaturation vapor pressure, determine the depth to which a particular icephase can be dried at a specific temperature. For amorphous ice at −160°C., the transition time is 205 days. Based upon extrapolation ofexperimentally determined saturation vapor pressures and the Knudsenequation, this would allow drying of 26 microns. At −140° C., transitiontime is 28 minutes and would allow drying of 0.8 μm under idealconditions. Below −160° C., i.e., prior to the onset of the transition,one could predict little, if any, translational kinetic energy of thewater molecules and hence little, if any, drying.

Based upon these considerations, one can postulate the hypothesis oftransitional drying, i.e., that for a sample containing multiple phasesof ice, it is possible to dry each phase sequentially during itstransition. The amount of each phase dried will obviously be dependentupon multiple parameters including efficiency of drying apparatus, rateof heating, and impedance of the dry shell.

Cryopreservation

Cryopreservation is the preservation of cell or tissue structure againstinjury associated with freezing events. Natural cryoprotection canresult from adaptive metabolism of the organism, with changes incellular structure, composition and metabolic balance giving an enhancedtolerance of freezing. In laboratory experiments when cell viability ortissue ultrastructure are to be preserved following cooling, two methodsare available. The first is to ultrarapidly cool the sample, resultingin the tissue fluids being vitrified, i.e., absence of ice crystals. Thesecond is to incorporate chemical additives to confer a degree ofcryoprotection. The chemicals range from naturally occurringcryoprotectants such as glycerol, proline, sugars, and alcohols toorganic solvents such as dimethylsulfoxide (DMSO) to high molecularweight polymers such as polyvinylpyrrolidone (PVP), dextran andhydroxyethyl starch (HES).

Vitrification of cells and tissues is limited by the rate at which thesample can be cooled and the insulating properties of the tissue itself.Due to physical limitations, one can only achieve vitrification of athin layer of tissues using state of the art techniques. This makes theidea of chemical additives for cryoprotection and manipulating thecooling rate very appealing in attempts to cool and store biologicalsamples without causing structural and functional damage.

Injury to biological samples due to freezing is subject to fundamentalphysical and biological principles, some long known, but others onlyrecently being understood. Serious investigations into the mechanisms offreezing injury in biological samples did not begin until the secondquarter of this century. These early studies were dominated by thebelief that physical damage by ice crystals was the principal cause offreeze injury. The effects of dehydration and a correlation between theconcentration of extracellular solutes and cell and tissue damage hasbeen demonstrated. A “two factor” hypothesis for cell freezing injuryproposed that cell injury was the result of either the concentration ofsolutes by extracellular ice or the formation of intracellular ice whichcaused mechanical injury.

The action of glycerol and other small polar compounds has beeninterpreted as penetrating and exerting colligative action within thecells. In the proportion that the colligative action of the penetratingcompounds maintains water in the liquid state at temperatures below 0°C., an increased volume of cellular solution is maintained. This avoidsan excessive concentration of toxic electrolytes in the nonfrozencellular solution. A similar influence also takes place in the externalsolution. In this context, colligative action is referred to as actionby an extraneous solute, in lowering the freezing point of the solutionin contact with ice. If enough protective compound is present, the saltconcentration does not rise to a critically damaging level until thetemperature becomes so low that the damaging reactions are slow enoughto be tolerated by the cells. Similar concepts of damage to tissuematrix by both mechanical growth of ice crystals and chemical damage dueto concentration of solute and changes in pH can also be applied.

The nonpenetrating cryoprotectants vary in size from sucrose to largepolymeric substances such as PVP, HES and dextran. It has been suggestedthat nonpenetrating substances act by some other means than that in thecolligative mechanism described above. The role of larger molecules isbelieved to be dehydrative by osmotic action. When a large proportion ofwater is withdrawn from the cells by means of an osmotic differential,less free water is available for intracellular ice crystallization whichis often identified as a lethal factor. In tissues, polymeric substancesmay act by binding and structuring water molecules.

The cooling rate in the presence of cryoprotective compounds is a veryimportant factor in freezing injury. Normally for cells, slow cooling isbetter than elevated cooling rates since the latter promotesintracellular ice formation. This occurs because there is insufficienttime for water to escape from the cells before the contained cell waterfreezes. With slow rate cooling, extracellular ice forms first,resulting in dehydration of the cell which, together with the presenceof the cryoprotectant, prevents intracellular ice formation. For tissuematrix samples there is a more direct correlation to the overallreduction in the degree of total ice crystal formation.

Penetrating compounds were thought to act by not allowing an excessivetransport of water from the cells too early in the freezing processwhile nonpenetrating compounds have a dehydrative effect on cells alongwith a colligative effect of diluting the solution surrounding the cell.Neither of these descriptions, however, tells the whole story.

Solutes such as HES and PVP are totally nonpenetrating, waterwithdrawing compounds of merely larger molecular weight thannonpenetrating sucrose. The larger molecular weight should render suchcompounds less osmotically and colligatively effective, when consideredon a weight basis. Yet in concentrated solutions, the compounds'colligative action has been shown to be far greater than would beexpected based on merely a linear relationship to concentration.

A source of damage to frozen tissue, other than freezing itself, is theosmotic and toxic effects of many of the cryoprotective agents. Whenused in mixtures, some cryoprotective compounds may counteract thetoxicity of other cryoprotectants, as was demonstrated by the additionof polyethylene glycol (PEG) to a mixture of DMSO and glycerol. Theinventors have developed several vitrification solutions (VS).

The toxicity of the individual components of these solutions weretested. In the mixtures, the toxic effects were lower than when anequivalent concentration of any one component was used alone. Theresulting solutions are nontoxic to cell cultures and remains glass likeand optically clear (i.e., no visible ice crystal is formed) whenplunged into liquid nitrogen. Vitrification Solution 1 Dimethylsulfoxide(DMSO)  0.5M Propylene glycol  0.5M 2-3 butanediol 0.25M Proline  1.0MRaffinose 2.5% (w/v) Polyvinylpyrrolidone (PVP)  15% (w/v) (Ave. M.W. ≈40,000) Dextran  15% (w/v) (Ave. M.W. ≈ 40,000-70,000)

A modified vitrification solution (VS₂) has also been developed whichcomprises a mixture of: DMSO  0.5M Propylene glycol  0.5M 2-3 butanediol0.25M Raffinose 10% (w/v) Trehalose  6% (w/v) Sucrose  6% (w/v) PVP 12%(w/v) (Ave. M.W. ≈ 40,000) Dextran 12% (w/v) (Ave. M.W. ≈ 40,000-70,000)

Another modified vitrification solution (VS₃) which has been developedcomprises a mixture of: DMSO  0.5M Propylene glycol  0.5M 2-3 butanediol0.25M Raffinose 2.5% (w/v) Sucrose  12% (w/v) PVP  15% (w/v) (Ave. M.W.≈ 40,000) Dextran  15% (w/v) (Ave. M.W. ≈ 40,000-70,000)

A fourth modified solution (VS₄) has been developed. This solutiondiffers in that it contains 50% formamide, an organic solvent. Thismixture neither expands nor contracts with freezing and hence does notcause cracking when freezing larger tissue samples. It comprises amixture of: Formamide 50% (w/v) 70K Dextran 15% (w/v) Raffinose 2.5%(w/v)  40K PVP 15% (w/v) Sucrose 12% (w/v)

In summary, the factors affecting the cryoprotective nature of compoundsare (a) chemical composition, (b) low toxicity, (c) molecular size andpenetrating ability, and (d) interaction with other compounds in themixture.

The physicochemical effects of cryoprotectants are (a) depression of theequilibrium freezing point of substrate and cytoplasm on a colligativebasis, (b) depression of homogeneous ice nucleation temperature, (c)reduced rate of ice crystal growth due to change in the viscosity andthermal diffusivity of the solution, and (d) dehydrative effects oncells by osmotic action.

Cooling Parameters

For purposes of cryopreparation of the biological tissues of thisinvention, it is essential to note that a variety of cooling processescan be used. In a preferred embodiment of this invention, rapid coolingis considered essential to obtain the proper ice crystal blend. In themost preferred embodiment of this invention, a vitrification procedureis used which results in the formation of a substantial proportion ofamorphous water in the biological sample. As will be disclosedhereinafter, regardless of the form of cooling that is used, it isbelieved that amorphous phase water, cubic ice crystals and hexagonalice crystals are present in the final product. The method of cooling hasa distinct bearing on the distribution of ice crystal types found in thecooled cryosolution.

Drying Parameters

The aim of controlled drying of a frozen biological tissue by moleculardistillation drying is to remove water from the sample without furthermechanical or chemical damage occurring during the drying process. Thisinvolves avoiding, by use of appropriate drying conditions, twofundamental damaging events. The first is to remove water from icecrystalline phases without transition to larger more stable and moredestructive crystals. The second is to remove water from solid butnoncrystalline water or water-solute mixtures without melting orcrystallization of these solid phases. This second component refers towater present in the amorphous condition, water together with solute inthe eutectic or water together with a compound which binds andstructures water and hence, prevents its crystallization during thefreezing process. Hence, vitreous water can be of low energy andstability, as in ultrarapidly-cooled pure water, or high energy andstability, as that achieved with cryoprotective agents with intermediaterates of cooling.

Many of the features required of controlled drying to avoid theoccurrence of these events are overlapping. The reason for this is thateach form of water will have a particular energy state, whether in acrystal or bound to a cryoprotective compound, and it is this energystate, rather than its configuration, which determines the requirementsfor drying. Consider for example, (1) a sample of cubic ice achieved bycooling pure water at an intermediate cooling rate and (2) vitrifiedwater achieved by mixing water with glycerol to 45% vol:vol and coolingat an intermediate rate. The first sample will be crystalline and theaim of drying is to remove water from this state without transition tohexagonal ice. The second sample is an amorphous solid and the aim ofdrying is to remove water from this phase without melting of the glassto a liquid with subsequent boiling. For cubic ice, the onset of itstransition is −130° C. and the rate of transition is temperaturedependent being very slow at −130° C. and very rapid at −90° C. For 45%glycerol-water, the glass transition temperature is −120° C. andrepresents the onset of melting. The melting process is very slow at−120° C. and is temperature dependent, becoming very rapid at −90° C.

Prior to the onset of the cubic to hexagonal transition or the glasstransition of 45% glycerol-water, the saturation vapor pressure of waterin these phases is extremely low and drying would occur at extremelyslow rates. The aim of controlled drying, therefore, is to remove waterfrom the cubic ice phase during its transition and in a time less thanis required for any significant transition to hexagonal ice and from the45% glycerol-water phase during its transition to a liquid but in lesstime than is required for any appreciable liquid to form.

This argument can be applied repetitively to all forms of water presentwhether it be crystalline in the form of cubic or hexagonal ornoncrystalline as amorphous or bound to any molecule, be itcryoprotectant, protein, carbohydrate, or lipid. To simplify thisconcept, water in a frozen biological sample can be described as havinga specific energy level E. In a frozen biological sample, there will bewater forms of multiple definable energy levels:

E₁ E₂ E₃ - - - E_(n)

The mode of preparation, the nature of the sample, the use ofcryoprotectants or other additives, and the cooling rate used willdetermine the relative proportions of these different water forms. Eachenergy level will determine the onset temperature of its transition ormelting and the temperature dependence of the rate of the transition ormelt.

Controlled drying processes must be able to remove each of thesedifferent states of water during the transition and in less time than isrequired to complete the transition. This mode of drying, therefore,requires that several conditions be met.

First, the frozen sample must be loaded into the dryer withouttemperature elevation above its lowest transition temperature. Ifelevation of temperature does occur, this must be over a short period oftime such that no appreciable transition occurs. Ideally, loading occursunder liquid nitrogen at −190° C., well below the lowest discernibletransition of −160° C. for pure, ultrarapidly-cooled amorphous water.If, however, the sample is predominantly cubic ice or a mixture of waterand cryoprotectants with a glass transition of the order of −100° C. to−130° C., a closed circuit refrigeration system may be sufficient toenable maintenance of the sample temperature below the onset oftransition.

Once loaded, the sample must be exposed to vacuum and be in direct lineof sight of the condenser surfaces. The criteria for these are againdetermined by the nature of the water phases present in the sample. Thefollowing objectives must be attained. The vacuum within the chamberduring the drying of a particular phase must create a partial pressureof water at least equivalent to or less than the saturation vaporpressure of water in the phase to be removed. This saturation vaporpressure is dependent on the nature of the water phase and itstemperature. Hence, for pure amorphous water in the transition range of−160° C. to −130° C., the approximate saturation vapor pressures are6×10⁻¹² mbar (−160° C.) and 5×10⁻⁷ mbar (−130° C.), respectively. As thetransition times of amorphous to cubic ice in this same temperaturerange, −160° C. to −130° C., vary from 5×10⁵ minutes to 5 minutes,drying will be very slow until temperatures of the order of −150° C. to−140° C. are reached requiring a vacuum of 5×10⁻¹⁰ to 2×10⁻⁸ mbar. Thisrepresents one extreme.

For cubic ice, little if any drying will occur below its onset oftransition at −130° C. as its saturation vapor pressure will be of theorder of one log lower than for amorphous water. In the transitionrange, −130° C. to −100° C., the saturation vapor pressure of cubic iceis approximately 5×10⁻⁸ to 9×10⁻⁵ mbar. The transition times of cubic tohexagonal are 700 minutes and 109 minutes respectively. The saturationvapor pressure, therefore, determines the vacuum requirements for dryingand can be applied to all water phases present. It is important to notethat the same vacuum criteria are not applicable to all phases, butrather are phase-dependent.

A second criteria of the vacuum is that the mean free path be in excessof the distance between the sample and the condenser surface. Ideally,this should be a tenfold excess. The condenser surface must be a lowertemperature than the onset transition temperature of the phase of waterbeing removed from the sample so that the saturation vapor pressure ofwater condensed on this surface during drying is considerably lower thanthat of the water phase within the sample. Ideally, this should be threeorders of magnitude lower. For a sample containing multiple waterphases, the temperature of the condenser surface must remain below theonset of transition of the least stable ice phase remaining to beremoved. Ideally, the condenser should also be in line of sight of thesample.

Once the sample has been loaded and exposed to vacuum and the condensersurfaces, the sample and sample holder must be heated so as to increasethe mobility of water molecules and hence, cause their escape. This isthe essential and critical component in the drying of a samplecontaining multiple phases or energy levels of water. The temperature ofthe sample must be accurately known. The control of temperature and therate of sample heating must be accurately controlled. This is necessaryto ensure that the drying of each phase of water in the sample issequential.

Hence, for a sample containing multiple phases of water of energy levelE₁, and E₂ - - - E_(n) where E₁ is the least stable, then heating mustoccur at such a rate that E₁ is removed prior to its transition to E₂.E₂ prior to its transition to E₃ and so on. This requires nonequilibriumdrying conditions and heating at a continuous rate or by holding at aconstant temperature level such that sublimation occurs as determinedby: ${Js} = {{NPs}( \frac{M}{2\quad\pi\quad{QT}} )}^{0.5}$where

-   -   Js=sublimation rate in g cm⁻¹ sec⁻¹    -   N=coefficient of evaporation    -   Ps=saturation vapor pressure    -   M=molecular weight of water    -   Q=universal gas constant    -   T=absolute temperature of the sample.

This is consistent with the transition rate for the particular phasebeing removed. For example, the rate of the amorphous to cubictransition is given by:E=2.04×10²⁸×exp(−0.465T)

Alternatively, if the transition window is T₁ to T₂, the sublimationrate and the transition rate will vary with temperature during thisinterval. The rate of heating during this window T₁ to T₂ must be suchthat sublimation occurs throughout the dimensions of the sample beforetransition at any particular temperature is completed.

In this way, the aim of controlled drying is achieved, i.e., thesequential removal of each phase of water under conditions appropriateto the properties of each phase without appreciable ice crystal growth,formation or melting of the particular phase. Once dry, the sample mustbe physically or mechanically isolated from water on the condensersurface or any other source and stored in a closed container eitherunder vacuum or dry inert gas.

In a preferred embodiment, samples are cooled by an appropriate methodsuch that ice crystal formation is below the degree that would causedamage to the sample. Once frozen, the sample is then stored below thetransition temperature of the most unstable ice form. For amorphous ice,this is preferentially below −160° C. The sample is then loaded into asample holder, precooled to −196° C. and transferred into a moleculardistillation dryer. The dryer chamber is then closed and sealed forvacuum integrity. To avoid recrystallization, the hydrated sample mustremain below the transition temperature of the most unstable ice formthroughout all manipulations.

Once the sample is loaded, high vacuum (10⁻⁸ to 10⁻⁶ mbar) is generatedinside the chamber. The sample is placed considerably closer to thecondenser surface (liquid nitrogen cooled chamber walls) than the meanfree path within the chamber. The condenser temperature must always bebelow that of the sample. For an amorphous sample, the condenser ispreferentially −196° C.

The sample holder is then heated via a programmable heatermicroprocessor thermocouple loop. Heating programs are determinedaccording to the ice composition of the sample. A typical program for asample containing amorphous, cubic and hexagonal ice is 10° C. per hourfrom −180° C. to −150° C., 1° C. per hour from −150° C. to −70° C., and10° C. per hour from −70° C. to +20° C.

Once the sample has reached 20° C., it can be sealed inside anappropriate container within the vacuum chamber and unloaded forsubsequent storage. In one configuration, the sample is contained withina glass vial and sealed with a butylrubber lyophilization stopper at theend of cycle. More specific details of the operation of the moleculardistillation dryer are given in U.S. Pat. No. 4,865,871.

Reconstitution

The freezing and drying of biological tissues impart great physicalstress upon the bonding forces which normally stabilize macromolecularconformation. Contributing to this destabilizing effect is the increasein concentration of electrolytes and possible pH changes as the solutionfreezes. As a consequence, modifications to the sample, including theinactivation of certain enzymes, and the denaturation of proteins, mayresult.

Studies with lactic dehydrogenase have shown that freezing and thawingcause dissociation of the tetrameric enzyme into subunits which isaccompanied by a change in biological activity. The dissociation wasfound to be dependent on the ionic strength and pH during freezing.

Other studies investigating the quaternary structure of L-asparaginasedemonstrated that this enzyme dissociated from the active tetramer toinactive monomers when freeze-dried. This monomeric state was found tobe stabilized by reconstitution of the dried enzyme with buffers of highpH and high ionic strength. However, the dissociation was shown to becompletely reversible on reconstitution at neutral pH and low ionicstrength. The effect of pH on the other hand may induce changes in thethree dimensional structure resulting in subunits conformationallyrestrained from reassociation.

These studies indicate the importance of determining optimal pH andionic strength conditions of not only the formulation used in thecryopreservation protocol, but also the reconstitution solution. In thisway, maximal sample activity and stability may be obtained.

Other variables of reconstitution such as vapor phase rehydration ortemperature may also be important to the retention of activity followingfreezing and drying. Other workers in the field have demonstrated amarked difference in proliferative response to lectins depending on thetemperature of rehydration or whether samples were reconstituted byvapor phase. Improved responses to lectins were noted when thefreeze-dried lymphocytes were rehydrated at dry ice temperatures andthen allowed to warm. This gradual method of reconstitution reduced theosmotic stress induced by sudden rehydration.

In the processing of biological tissues, the rehydration step can alsobe used to augment the processing and stabilization compounds used inthe procurement and processing steps. These include components tominimize the effects of hypoxia and free radical generation, agents toinhibit enzymes, oncotic agents including proteoglycans, dextran andamino acids to prevent osmotic damage.

In addition, the rehydration of certain tissues, e.g., the vascularconduits and heart valves, may require specific agents to inhibitplaletet aggregation during the early post implant period. Where thebiological tissue is to be crosslinked, rehydration directly in thefixative has the additional advantage of immediate and uniformdistribution of the fixative throughout the tissue.

Storage Considerations

Sublimation of water from a frozen sample is one method for preservingthe active components of biological material. However, the optimalpreservation of activity with long-term stability requires criticalcontrol of the drying process and storage conditions. Following theremoval of free or unbound water, the process of secondary dryingproceeds, during which structurally bound water is removed. Bound wateris intimately associated with the maintenance of protein conformation.Thus, the amount of water remaining in the dried sample, known as theresidual moisture content, is a significant variable in the dryingprocess. The final residual moisture content affects both the survivaland stability of the sample.

Residual moisture content is expressed as the “percentage residualmoisture” and is equated to the weight (gm) of residual water per unitweight (gm) of original sample.

It is generally agreed that biological materials dried by vacuumsublimation of ice show increased stabilization when dried to optimumcontents of residual moisture. Materials which have been under oroverdried, i.e., to moisture contents that are above or below theoptimum, will show increased deterioration.

Although the optimal residual moisture content will vary depending onthe particular dried sample, certain stability problems can be expectedwhen the levels of moisture are suboptimal. Overdrying a sample, i.e.,residual moisture contents less than 1-2% without using a drystabilizer, generally results in removal of nearly all structured waterallowing modification or blocking of exposed hydrophilic sites ofproteins by oxidation. This oxidation causes degradation with acorresponding decrease in the biological activity. On the other hand,residual moisture contents of greater than 5% generally are indicativeof underdrying where sufficient amounts of “free water” remain in thesample which could contribute to transconformation of the protein. Theresulting rearrangements of the polypeptide chains shift from thetypical ordered arrangement of the native protein to a more disorderedarrangement. These protein perturbations can result in poor long-termstability of the dried product.

Successful long-term storage requires sample drying to optimal levels ofresidual moisture. Inadequate drying of biological samples and itsconsequences have been shown in the literature. Maximal stability ofsuspensions of influenza virus dried by sublimation of water in vacuooccurred at a residual moisture content of approximately 1.7%. Under orover drying to non-optimal water content resulted in the degradation ofthe virus suggesting that varying amounts of free and bound water in adried sample have an effect upon protein structure and activity.

To maximize sample stability and satisfy regulatory requirements for thepreparation of dried pharmaceuticals or reagents, it is essential thatthe residual moisture content be determined following sample drying.

Several methods are available to measure residual moisture contents;

-   -   1. Gravimetric (Heating Method)—A known quantity of dried        product is heated and the weight loss can be equated with water        content.    -   2. Chemical Assay—This method is based on the reaction between        water and free iodine in a mixture of pyridine, sulphur dioxide        and methanol. The endpoint is detected coulometrically when free        iodine is present. H₂O+I₂+SO₂+ROH+3RN→2RNHI+RN+HSO₄R    -   3. Gas Chromatography

Each of the methods has limitations and therefore, it is wise to notrely on any single method of moisture determination. Rather, multiplemethods should be employed to validate the results.

Once dried to optimal residual moisture contents, the sample is stillconsidered unstable when removed from the vacuum due to its hygroscopicnature and susceptibility to oxidation. Measures must be taken duringstorage to protect the sample from atmospheric rehydration and minimizeexposure to oxygen. Such protection is essential to the maintenance ofthe sample's long-term stability.

Evidence in the literature indicates that the gaseous condition underwhich the samples are sealed, as well as the storage temperature,effects the long-term stability of the sample. It has been demonstratedin a study comparing different gases and storage temperatures, thatmaximum stability of influenza virus was obtained when samples werestored under helium or hydrogen gas at low temperature (−20° C.).Sealing under other gases or vacuum at different storage temperaturesresulted in varying levels of stability. The inventors postulate thatthose conditions which most effectively limit oxygen contact with thesample, markedly improve biological activity by reducing oxidation ofexposed hydrophilic sites at the protein surface. Appropriate storageparameters, i.e., temperature, and sealing under gas or vacuum areimportant to obtain long-term sample stability.

EXAMPLE 1 Processing and Storage of Transplantable Skin

Human donor skin is routinely harvested from cadavers and stored underrefrigerated or frozen conditions at a number of tissue banks throughoutthe nation. This skin is used as a temporary dressing for burn victimsthat are undergoing extensive autografting. Porcine skin is alsoharvested under similar conditions and used as a temporary burndressing. In its unprocessed condition, the allogeneic skin and porcineskin are ultimately rejected by the patient. This same skin is alsoavailable for processing by the methods described below.

Donor skin is harvested under aseptic conditions with a dermatome, andmaintained at 4° C. in RPMI 1640 tissue culture media containingpenicillin and streptomycin solution for no more than 7 days prior tofurther processing. Transportation to LifeCell's tissue processingcenter is via overnight delivery, on wet ice, in the same media. Onarrival at the processing center, the temperature of the tissuecontainer is verified to be at least 4°, or the skin discarded.Following verification of container temperature, donor identificationand test screening data, the skin is transferred to a laminar-flow hoodfor further processing.

The donor skin is removed from the transportation container and placedwith its reticular side down on a piece of sizing support being a lowdensity polyethylene. An appropriately sized piece of gauze is added tothe epidermal side of the skin which is then cut into a rectangularpiece as large as possible, not to exceed a 4×4 inch square and nosmaller than 2×3 inches. The skin is then placed reticular side down, ina petri dish, to which 50 ml of De-epidermizing Solution consisting of1M NaCl is added. The petri dish is then transferred to an incubator andincubated at 37°±2° C. for 18 to 32 hours for human skin and 35 to 55hours for porcine skin.

After incubation, the petri dish containing the skin is transferred to alaminar flow hood for deepidermization. The gauze is first removed anddiscarded. The epidermis is then gently grasped with forceps and pulledaway from dermis as a sheet. The excess Deepiderizing Solution is thenaspirated. A slit approximately one centimeter long is then made in thelower left corner of the dermis to identify the upper and lowersurfaces.

The dermis is next rinsed in the same petri dish by the addition of 50ml Tissue Wash Solution, consisting of sterile Hanks balanced saltsolution. The petri dish is then placed on a rotator at 40±5 RPM for 5minutes at room temperature (20°-26° C.). The petri dish is thenreturned to the laminar flow hood and the lid from the petri dish isremoved in order to aspirate the Tissue Wash Solution. This procedure isrepeated a further two times.

The dermis is then treated with 50 ml. of De-Cellularizing solution andthe petri dish is placed on a rotator at 40±5 RPM for 1 hour at roomtemperature (20°-26° C.). The decellarizing solution for human skinconsists of 0.5% sodium dodecyl sulfate in Hanks balanced salt solutionand for porcine skin contains 1 mM disodium ethylenediamine tetraaceticacid (EDTA). The De-cellularizing solution is removed by aspiration. Thedermis is then washed with 50 ml of Tissue Wash Solution. The petri dishis then placed on a rotator at 40±5 RPM for 5 minutes at roomtemperature (20°-26° C.). The Tissue Wash Solution is removed byaspiration. The washing procedure is repeated (2) times. After thedermis has been washed a total of 3 times 50 ml of Pre-freezing Solutionis added to the petri dish. The dish is then placed on a rotator at 40±5RPM for 30 minutes at room temperature (20°-26° C.). The prefreezingsolution for human skin consists of 7% dextran (70,000 MWT), 6% sucrose,6% raffinose and 1 mM disodium ethylenediamine tetraacetic acid in Hanksbalanced salt solution. The prefeezing solution for porcine skinconsists of 7.5% dextran (70,000 MWT), 6% sucrose, 7.5%polyvinylpyrrolidone (MWT 40,000), 1.25% raffinose and 1 mM disodiumethylenediamine tetraacetic acid made up in Hanks balanced saltsolution.

A new piece of gauze is then placed on the papillary side of the dermisand the dermis is turned over so that the reticular side faces up. Thebacking from the reticular side of the piece of dermis is discarded intoa biohazard waste container. An approximately 0.5 to 1.0 cm wide stripof backing and dermis is then cut from the original sample. This stripis then cut into two satellite pieces, each approximately 1.0 cm long.All necessary quality assurance is ultimately performed on thesesatellite samples, including microbiology and structural analysis.

The tissues are then transferred into individual Tyvec bags. The tissuesare positioned in the bag backing side up with the white vent side down.The Tyvec bag is then heat sealed.

The sealed Freeze-dry Bag is transferred to a freeze-dryer which has aminimum shelf temperature of −70° C. and a minimum condenser temperatureof −85° C. The tissue is then frozen on the freeze-dryer shelf byramping the shelf temperature at a rate of −2.5° C./minute to −35° C.,and held for at least 10 minutes.

The drying cycle is such that the final residual moisture content of thesample is less than 6% and optimally 2%. In this example, the frozendermis is dried by the following program:

-   -   1. The shelf temperature is ramped at a rate of −2.5° C./minute        to −35° C., and held for 10 minutes, with vacuum set to 2000 mT.    -   2. The shelf temperature is then ramped at a rate of 1.5°        C./minute to −23° C., and held for 36 hours with vacuum set to        2000 mT.    -   3. The temperature is then ramped at rate of 1.5° C./minute to a        shelf temperature of −15° C., and held for 180 minutes with        vacuum set to 2000 mT.    -   4. The temperature is then ramped at a rate of 1.5° C./minute to        a shelf temperature of −5° C. and held for 180 minutes with        vacuum set to 2000 mT.    -   5. The temperature is finally ramped at a rate of 1.5° C./minute        to a shelf temperature of 20° C. and held for 180 minutes with        the vacuum set to 0 mT.

Following drying, the Freeze-dry Bag containing the dried dermis isunloaded under an atmosphere of dry nitrogen gas, placed in a secondpredried impervious pouch and heat sealed under the same inertenvironment.

(During the processing procedure and prior to sealing for freeze drying,a satellite sample is cut from the main sample and further processedunder identical conditions to the main sample. Prior to use of the mainsample in transplantation, all necessary quality assurance is performedon the satellite sample, including microbiology and structuralanalysis.)

Following drying, the sample is stored at above freezing temperatures,optimally 4° C. in a light protected environment.

Prior to use, the sample is removed from the sealed pouch under asepticconditions and rehydrated by immersion in balanced salt solution at 20°to 37° C. Rehydration is complete after 30 minutes of incubation in thisrehydration solution.

Analysis of the end product by light and electron microscopy hasdemonstrated it to be structurally intact with normal collagen bandingand the presence of collagen bundles in the matrix of the dermis andwith structural preservation of the lamina densa and anchoring fibrilsof basement membrane complex.

The reticular aspect of processed dermis has been demonstrated toprovide a substratum for the outgrowth of keratinocytes from a foreskinexplant in a laboratory by cell culture methods. The processed dermishas also been demonstrated to support the growth of isolatedkeratinocytes. In this circumstance, when cultured at an air liquidinterface, keratinocytes differentiate to all identifiable layers ofnormal skin and interact with the processed dermis through the basementmembrane complex. Processed porcine skin has also been demonstrated tosupport the growth of keratinocytes from human foreskin explants.

The processed dermis, either in combination with a meshed, ultra thin orepidermal autologous graft or reconstituted with cultured keratinocytes,has a number of clinical applications in full thickness skin injury.These include, but are not limited to, burn patients, patients sufferingfrom venous, diabetic, or pressure ulcers, and patients who undergoreconstructive surgery, or skin replacement following excision of skinlesions.

Processed human and porcine skin have been shown to undergo fibroblastinfiltration and neovascularization in human burns patients and insurgically induced full thickness skin injury in pigs.

EXAMPLE 2 Vascular Conduit: Human Donor Saphenous Veins

Saphenous veins are harvested from cadaver donors and made available bytissue banks across the U.S. Tissue banks have established procurementguidelines, published by the American Association of Tissue Banks. Theseguidelines include instructions for patient selection, completion ofconsent forms and a caution to avoid mechanical distention or othermechanical damage to the vein during the dissection process.

Harvesting begins with flushing and distension of the vein with VeinFlushing Solution, consisting of 1000 cc PlasmaLyte Solution forinjection, amended with 5000 units of Heparin and 120 mg of Papaverine(1 liter per vein). The veins are carefully removed under sterileconditions with as many tributaries maintained intact as possible, witha length of at least 5 mm. These tributaries are ligated with 3-0 silk.The surrounding fatty tissue is also maintained with wide margins aroundthe vein. Once the vein is removed, it is rinsed again with VeinFlushing Solution, packaged in 500 cc of cold (4° C.) Vein TransportMedium, consisting of 500 cc RPMI 1640 Tissue Culture Medium amendedwith 60 mg Papaverine and shipped by overnight delivery to a tissue bankfor further processing.

At the tissue bank, all tributaries are suture ligated and thesubcutaneous fat/soft tissue removed using standard surgical procedures.Following dissection, the vein is disinfected of any surfacecontaminants by placing it in a tissue culture medium amended withCefoxitin (240 mcg/ml), Lincomycin (120 mcg/ml), Polymyxin B Sulfate(100 mcg/ml) and Vancomycin (50 mcg/ml). The vein is maintained in theantibiotic mixture at 4° C. for 24 hours. The disinfected vein is placedin 500 cc of cold (4° C.) Transport Medium, consisting of 500 cc RPMI1640 Tissue Culture Medium and transported on wet ice to LifeCell'sTissue Processing Center by overnight delivery.

On arrival, the container temperature is verified to be at least 4° C.Following verification, the vein is placed into a container containingCryosolution and incubated for one hour at room temperature. TheCryosolution consists of the following:

0.5M Dimethyl Sulfoxide (DMSO)

0.5M Propylene Glycol

0.25M 2-3 Butanediol

2.5% (w/v) Raffinose

12.0% (w/v) Sucrose

15.0% (w/v) Polyvinylpyrrolidone (PVP)

15.0% Dextran.

After incubation, the vein is then placed into an inert plastic bagcontaining a porous vent which allows water vapor to pass out, butprevents bacteria from passing in and is heat sealed. The bag and veinis then frozen by plunging into liquid nitrogen. The frozen vein isstored at temperatures below −160° C.

For drying, the frozen vein within the bag is transferred under liquidnitrogen to a molecular distillation dryer, and dried by methodsdescribed in U.S. Pat. No. 4,865,871. For saphenous veins processed inthe above described cryosolution and rapidly frozen, the optimum rangefor drying is −130° C. to −70° C. with a heating rate of 1° C. perminute during the drying phase. Once dry, the vein is sealed in thecontainer under dry inert nitrogen gas and stored at refrigeratedtemperatures (2-4° C.) until needed for transplantation.

The vein is rehydrated in a vapor phase, by opening the plastic pouchcontainer and placing the vein in a 37° C. humidified incubator. Thevein is maintained in this incubator for one hour, after which it isremoved and placed in a container with phosphate buffered saline (PBS).The vein is then rinsed with 3 changes of PBS.

Analysis of the processed veins show them to possess an intactextracellular matrix both by light and electron microscopy. Proteasedigestion indicates no increased susceptibility of collagen todegradation. Stress testing on a dynamic loop with an artificial hearthas demonstrated them to withstand supraphysiological pressures withoutcompromise of their leak barrier function to either liquid or gas.

EXAMPLE 3 Vascular Conduit Processing for Animal Study

Procurement

Twenty to thirty kilogram mongrel dogs of either sex are induced viasodium pentathol, intubated, and prepped and draped in a sterilefashion. Anaeshesia is maintained with oxygen, nitrogen, and Halothane.A midline incision is made in the neck whereupon the external jugularveins and internal carotid arteries are exposed, isolated, and freed ofsurrounding fascia. During this procedure, a flushing solution comprisedof 5000 units of heparin and 120 mg of Papavarine in 1000 cc sterileHank's Buffered Saline Solution (HBSS) of pH 7.4 is sprayed on thevessels via a needle and syringe. The proximal and distal ends of thevessel are then clamped with atraumatic vascular clamps whereupon thevessel is rapidly excised. Immediately the vessel is flushed through andthrough with the above mentioned flushing solution and placed in 4° C.flushing solution for transport. Alternatively, the vessel may be placedin the below mentioned Decellularization Solution A for incubationduring transport.

Decellularization

After the trimming of any excess fascia, the vessel is placed inDecellularization Solution A (DSA). DSA is comprised of 25 mM EDTA, 1 MNaCl, and 8 mM CHAPS or similar zwitterionic detergent in a sterile PBSbase at 7.5 pH. After a 30 minute to one hour incubation, the vessel isgiven two ten minute washes in PBS and then placed in DecellularizationSolution B (DSB). DSB is comprised of 25 mM EDTA, 1 M NaCl, and 1.8 mMSodium Dodecylsulfate (SDS) or similar anionic or nonionic detergent ina sterile PBS base at 7.5 pH. After a 30 minute to one hour incubation,the vessel is given two ten minute washes in PBS.

Vitrification

After decellularization, the vessel is placed in Vitrification SolutionFifty-fifty (VSFF) for one to five hours. VSFF is comprised of 2.5%raffinose, 15% polyvinylpyrrolidone (PVP) of 40,000 molecular weight,15% Dextran of 70,000 molecular weight, and 12% sucrose in a 50/50 (byvolume) water-formamide solution. The vessel is then rapidly submergedin liquid nitrogen (LN₂) until frozen as evidenced by the cessation ofboiling. The vessel may then be stored in LN₂ or LN₂ vapor, orimmediately dried.

Drying

After vitrification, the vessel is transfered in a nitrogen gasatmosphere to a special Molecular Distillation Dryer sample holder whichhas been pre-cooled to −196° C. The sample holder is then rapidlytransferred under nitrogen gas atmosphere to the Molecular DistillationDryer. The dryer is then evacuated and run according to a protocoldeveloped specifically for VSFF. Under a vacuum less than 1×10⁻⁶ mbar,the sample holder is warmed according to the following protocol:

-   -   −196° C.->−150° C. over 10 hours    -   −150° C.->−70° C. over 80 hours    -   −70° C.->20° C. over 10 hours

The dryer is then opened and the vessel is transferred to a sealedsterile glass vial under nitrogen gas atmosphere. The vessel is thenstored at 4° C. until needed.

Rehydration

Twenty-four hours prior to use, the glass vial is opened in a 100%humidity, 37° C. atmosphere. The vessel is allowed to vapor rehydrate inthis manner for one to two hours. The vessel is then submerged insterile PBS at 4° C. for two hours. The PBS is then exchanged with freshsolution whereupon the vessel is stored at 4° C. overnight. The vesselis ready for use the following day.

EXAMPLE 4 Porcine Heart Valve Leaflets

Porcine heart valves were obtained from isolated hearts immediatelyfollowing slaughter at an abattoir. Discs from the leaflets of theintact valve were obtained by punch biopsy under aseptic conditions andtransferred to a transportation solution comprising Dulbecco's PBS with5.6 mM glucose, 0.33 mM sodium pyruvate with added anti-oxidantscomprising 0.025 mg/l alpha-tocophenol phosphate, 50 mg/l ascorbic acidand 10 mg/l glutathione (monosodium) at 4° C.

Upon receipt of tissue, the discs were transferred to a cryosolutioncomprising 0.5 M DMSO, 0.5 M propylene glycol, 0.25 M 2-3 butanediol,2-5% raffinose, 15% polyvinyl pyrrolodone, 15% Dextran and 12% sucroseand incubated at 20° C. for 60 minutes with moderate agitation.

Tissue samples were then placed on thin copper substrates matching thesize of the tissue sample and cooled by immersion in liquid nitrogen.

The frozen samples were then stored at below −160° C. until furtherprocessing.

Prior to drying, the samples were transferred under liquid nitrogen to asample holder equipped with thermocouple and heater. The sample holderwas precooled to liquid nitrogen temperature, and the transfer wascompleted under liquid nitrogen.

The frozen samples were then loaded into a molecular distillation dryerand dried by molecular distillation drying employing the methoddescribed in U.S. Pat. No. 4,865,871. The drying cycle employed was−180° C. to −150° C. in 3 hours, −150° C. to −70° C. in 80 hours and−70° C. to +20° C. in 9 hours. Following drying, the vacuum in thedrying chamber was reversed with ultrapure nitrogen gas and the discsmaintained in this atmosphere until processing.

Rehydration of the dry samples first consisted of exposure of samples to100% humidity at 37° C. for 60 minutes. Samples were then rehydrated ina rehydration solution which consisted of one of the following:

-   -   a. 0.06 M Hepes buffer    -   b. 0.06 M Hepes buffer+0.06 M MgCl₂    -   c. 0.06 M Hepes buffer+1% SDS    -   d. 0.06 M Hepes buffer+0.5 mM PMSF

Samples were incubated with agitation for at least four hours.

Following rehydration, samples were assessed under the followingcriteria:

-   -   a. Structure was assessed by both light and electron microscopy        and the valve matrix was found to be indistinguishable from that        of fresh unprocessed samples.    -   b. Protease digestion was found to be equivalent to fresh        sample.    -   c. Stress testing (static) was found to be able to withstand        greater stress load than control samples.    -   d. Subcutaneous animal implant model with subsequent explant at        7 or 21 days.        Explanted samples demonstrated:    -   i. Decreased capsule formation relative to fresh or        cryopreserved controls    -   ii. Decreased calcification relative to glutaraldehyde treated        controls    -   iii. Variable inflammatory cell infiltration depending on the        nature of the rehydration solution as follows:        Treatment: 0.06 M MgCl₂ in 0.06 M Hepes buffer

Clearly demarcated disc with well defined normal valve morphology.Sample incompletely surrounded by thin capsule with minimal inflammatorycell infiltration near disc periphery.

Treatment: 1% SDS in 0.06 M Hepes buffer

Clearly demarcated disc with well defined normal valve morphology.Sample completely surrounded by a slightly thicker capsule than thatobserved in MgCl₂ treated sample. Minimal inflammatory cellinfiltration.

Treatment: 0.5 mM PMSF in 0.06 Hepes buffer

Well defined normal valve morphology. Capsule formation nearly absent.Minimal inflammatory cell infiltration.

Treatment: Control—0.06 M Hepes buffer

Poorly defined valve structure. Massive inflammatory cell infiltration,but little evidence of capsule formation.

EXAMPLE 5 Intact Porcine Heart Valves

Procurement

Porcine heart valves are obtained from isolated hearts immediatelyfollowing slaughter at an abattoir. The aortic valve and at least oneinch or more of ascending aorta is then carefully excised withpre-sterilized instruments.

The valve is washed twice in sterile phosphate buffer solution (PBS) andthen placed in sterile, 10° C. PBS for transport. Within three hours ofprocurement, the valve is brought to the LifeCell facility where it isfurther trimmed and processed.

Decellularization

After trimming, the intact valve is placed in Decellularization SolutionA (DSA). DSA is comprised of 25 mM EDTA, 1 M NaCl, and 8 mM CHAPS orsimilar zwitterionic detergent in a sterile PBS base at 7.5 pH. After a30 minute to one hour incubation, the valve is given two ten minutewashes in PBS and then placed in Decellularization Solution B (DSB). DSBis comprised of 25 mM EDTA, 1 M NaCl, and 1.8 mM Sodium Dodecylsulfate(SDS) or similar anionic or nonionic detergent in a sterile PBS base at7.5 pH. After a 30 minute to one hour incubation, the valve is given twoten minute washes in PBS.

Vitrification

After decellularization, the valve is placed in Vitrification SolutionFifty-fifty (VSFF) for one to five hours. VSFF is comprised of 2.5%raffinose, 15% polyvinylpyrrolidone (PVP) of 40,000 molecular weight,15% Dextran of 70,000 molecular weight, and 12% sucrose in a 50/50 (byvolume) water-formamide solution. The valve is then rapidly submerged inliquid nitrogen (LN₂) until frozen as evidenced by the cessation ofboiling. The valve may then be stored in LN₂ or LN₂ vapor prior todrying.

Drying

After vitrification, the valve is transfered in a nitrogen gasatmosphere to a special Molecular Distillation Dryer sample holder whichhas been pre-cooled to −196° C. The sample holder is then rapidlytransferred under nitrogen gas atmosphere to the Molecular DistillationDryer. The dryer is then evacuated and a heating cycle initiated whichhas been optimized specifically for dehydration of VSFF. Under a vacuumless than 1×10⁻⁶ mbar, the sample holder is warmed according to thefollowing protocol:

-   -   −196° C.->−150° C. over 10 hours    -   −150° C.->−70° C. over 80 hours    -   −70° C.->20° C. over 10 hours

The dryer is then opened and the valve transferred to a sealed sterileglass vial under nitrogen gas atmosphere. The valve is then stored at 4°C. until required for transplantation.

Rehydration

Twenty-four hours prior to use, the glass vial is opened in a 100%humidity, 37° C. atmosphere. The valve is allowed to vapor rehydrate inthis manner for one to two hours. The valve is then submerged in sterilePBS at 4° C. for two hours. The PBS is then exchanged with freshsolution whereupon the valve is stored at 4° C. overnight. The valve isready for use the following day.

While the invention has been described in terms of the preferredembodiments, it will be apparent to those of skill in the art thatvariations and modifications may be applied to the compositions, methodsand in the steps or in the sequence of steps of the methods describedherein without departing from the concept, spirit and scope of theinvention. Such substitutes and modifications are considered to bewithin the scope of the invention as defined by the appended claims.

1. A method of making a tissue matrix, the method comprising: (a)procuring a collagen-based tissue; and (b) incubating the collagen-basedtissue in a processing solution to produce processed tissue, wherein theprocessing solution extracts viable cells from the structural proteinand collagen matrix of the collagen-based tissue, wherein the processedtissue comprises a basement membrane to which viable endothelial cellsor viable epithelial cells securely attach.
 2. The method of claim 1,further comprising the steps of: (c) cryopreparing the processed tissueby incubation in a cryoprotective solution; and (d) freezing theprocessed tissue to produce a frozen, processed tissue, wherein thefrozen, processed tissue comprises a basement membrane to which, afterthawing of the frozen, processed tissue, viable endothelial cells orviable epithelial cells securely attach.
 3. The method of claim 2,further comprising the step of: (e) drying the frozen, processed tissueto produce a dried, processed tissue, wherein the dried, processedtissue comprises a basement membrane to which, after rehydration of thedried, processed tissue, viable endothelial cells or viable epithelialcells securely attach.
 4. The method of claim 3, further comprising thestep of: (f) incubating the dried, processed tissue in a rehydrationsolution to give a rehydrated tissue, wherein the rehydrated tissuecomprises a basement membrane to which viable endothelial cells orviable epithelial cells securely attach.
 5. The method of claim 4,further comprising the step of: (g) inoculating the rehydrated tissuewith autogeneic cells, allogeneic cells, or a combination of autogeneiccells and allogeneic cells, wherein the rehydrated tissue, after theinoculation, comprises a basement membrane to which viable endothelialcells or viable epithelial cells securely attach.
 6. The method of claim1, wherein the collagen-based tissue comprises dermis.
 7. The method ofclaim 1, wherein the collagen-based tissue comprises a tissue of venousor arterial origin.
 8. The method of claim 7, wherein the tissue ofvenous origin is saphenous vein tissue.
 9. The method of claim 1,wherein the collagen-based tissue comprises a heart valve.
 10. Themethod of claim 1, wherein the collagen-based tissue comprises heartvalve leaflet tissue.
 11. The method of claim 1, wherein thecollagen-based tissue is a human collagen-based tissue.
 12. The methodof claim 1, wherein the collagen-based tissue is a non-human animalcollagen-based tissue.
 13. The method of claim 12, wherein the non-humananimal is a pig.
 14. The method of claim 12, wherein the non-humananimal is a dog.
 15. The method of claim 1, wherein the collagen-basedtissue comprises a tissue selected from the group consisting of ligamenttissue, tendon tissue, bone tissue, cartilage tissue, dura mater tissue,and nerve tissue.
 16. A tissue matrix made by the method of claim
 1. 17.A method of treatment comprising grafting the tissue matrix of claim 16to or into a patient.
 18. The method of claim 17, wherein the patient isselected from the group consisting of a burn patient, a diabeticpatient, a patient with pressure ulcers, a patient requiringreconstructive surgery, and a patient requiring skin replacementfollowing excision of a skin lesion.